A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the β-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine²⁸ and glutamate⁸⁶, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.     

Identification of a new mutation in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

A mutation involving an A-to-G nucleotide replacement at position 985 of the medium-chain acyl-CoA dehydrogenase (MCAD) cDNA was found in homozygous form in 18 unrelated MCAD-deficient families and in heterozygous form in 4 families. By PCR amplification and sequencing of cDNA from a compound heterozygote, we have detected a new mutation in an MCAD-deficient patient in whom one MCAD allele produces mRNA that is missing 4 bp in the MCAD cDNA, while the other allele carries the A-to-G-985 mutation. The presence of this 4-bp deletion was confirmed in the patient's genomic DNA by dot-blot hybridization with allele-specific oligonucleotide probes and by restriction analysis of PCR products. A rapid screening test for this 4-bp deletion was developed, based on mismatched primer PCR amplification. The deletion created a new restrictive-enzyme site which yielded two DNA fragments. The 4-bp deletion was not found in the three remaining MCAD chromosomes not harboring the A-to-G-985 mutation, nor it was present in 20 chromosomes from 10 unrelated normal Caucasians. The PCR-based method for screening these two mutations can detect over 93% of all MCAD mutations.     

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.     

Immunoreactive enzyme protein in medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of mitochondrial fatty acid oxidation. To determine if immunoreactive enzyme protein is present in patients with MCAD deficiency, we studied cultured skin fibroblasts from patients with the 985 point mutation, present in about 85% of cases, and cell lines from patients in which the point mutation is not present or only involves one allele. Immunoblotting studies, using a polyclonal antibody to the purified protein, showed an absence of immunoreactive protein in mitochondrial fractions prepared from fibroblasts from MCAD-deficient patients. To determine whether MCAD protein accumulated in the cytosol because of impaired transport into the mitochondria, we immunoprecipitated MCAD protein from the fibroblast homogenate. MCAD protein was detected in the immunoprecipitates from controls, but not in those from the MCAD-deficient patients. These results suggest that either the MCAD protein is not synthesised or, if produced, it is rapidly degraded.     

Three RFLPs defining a haplotype associated with the common mutation in human medium-chain acyl-CoA dehydrogenase (MCAD) deficiency occur in Alu repeats.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of fatty-acid oxidation and may cause sudden infant death. Previous studies revealed that (i) homozygosity for an A-to-G mutation at nucleotide 985 of the mRNA coding region (A985G) is an extremely common cause of MCAD deficiency and (ii) MCAD deficiency is strongly associated with a particular haplotype for RFLPs for BanII, PstI, and TaqI. TaqI allele 2 is always associated with the A985G mutation in human MCAD deficiency. In this study, we have delineated the molecular basis of the RFLPs for PstI, BamHI, and TaqI in the human MCAD gene. Our results prove that the three RFLPs are caused by point mutations in the 8 kb of DNA encompassing exons 8-10 of the human MCAD gene. The TaqI polymorphism is caused by a C-to-A substitution 392 bp upstream of the exon 8, and the PstI and BamHI polymorphisms are due to T-to-C and G-to-A substitutions, respectively, which are 727 and 931 bp downstream of exon 10 respectively. All three RFLPs lie within Alu repetitive sequences. Comparison of intronic sequences immediately following exon 10 from two normal individuals with different haplotypes showed that this region contains densely packed Alu repeats and is highly polymorphic. Our results are consistent both with a founder effect as the cause of the high prevalence of a single (A985G) mutation in MCAD deficiency and with its association with a particular haplotype for these intragenic RFLPs.     

Prevalence of carriers of the most common medium-chain acyl-CoA dehydrogenase (MCAD) deficiency mutation (G985A) in The Netherlands

The G985A mutation represents about 90% of all medium-chain acyl-CoA dehydrogenase (MCAD) allele mutations that cause the clinical symptoms of MCAD deficiency. The prevalence of carriers varies between different European populations, with high frequencies in the northwestern part of Europe. To determine the prevalence of MCAD carriers with the G985A mutation in The Netherlands, we collected 6195 Guthrie cards of newborns. Mutation detection was performed with the polymerase chain reaction (PCR), in which a NcoI restriction site was created in the presence of a G985A mutation in the PCR product, followed by NcoI digestion, and gel electrophoresis. We detected a G985A carrier frequency of 1 in 59 (95% CI 1/50–1/73) in The Netherlands. The total prevalence of carriers was estimated to be 1 in 55 (95% CI 1/46– 1/68), based on a relative G985A frequency of 94% in The Netherlands. Received: 18 December 1995 / Revised: 14 February 1996     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Most cases of medium-chain acyl-CoA dehydrogenase deficiency escape detection in France

DNA from 414 French blood donors from the Paris area was assessed for the A985G mutation responsible for most cases of autosomal recessive medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. The mutant gene frequency averaged 1/140, predicting a frequency of mutant homozygotes of 1/19 000. Discrepancy between the numbers of expected (42 per year) and recorded cases of MCAD (6 per year) suggests that most MCAD-deficient patients escape detection in France.     

Disease-causing Mutations in Exon 11 of the Medium-Chain Acyl-CoA Dehydrogenase Gene

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of the mitochondrial β-oxidation in humans. It is a potentially fatal, autosomal recessive inherited defect. Most patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985), causing a change from lysine to glutamate at position 304 (K304E) in the mature MCAD. Only seven non-G985 mutations, all of which are rare, have been reported. Because the G985 mutation and three of the non-G985 mutations are located in exon 11, it has been suggested that this exon may be a mutational hot spot. Here we describe the results from sequence analysis of exon 11 and part of the flanking introns from 36 compound heterozygous patients with MCAD deficiency. We have identified four previously unknown disease-causing mutations (M301T, S311R, R324X, and E359X) and two silent mutations in exon 11. Our results show that exon 11 is not especially mutation prone. We demonstrate that two of the identified disease-causing mutations can be detected by restriction enzyme digestion of the PCR product from the assay for the G985 mutation, a discovery that makes this assay even more useful than before. On the basis of expression of wild-type and mutant MCAD protein in COS-7 cells, we show that the identified mutations abolish MCAD enzyme activity and that they therefore must be disease causing. The M301T, S311R, and K304E mutations are located in helix H, which makes up part of the dimer-dimer interface of the MCAD tetramer. On the basis of the three-dimensional structure of MCAD and the results from the COS-7 expression experiments, we speculate that the primary effect of the M301T and S311R mutations is on correct folding/tetramer assembly, as it has previously been observed for the K304E mutation.     

Origin of Finnish mutations causing aspartylglucosaminuria

Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disease highly enriched in Finland where one mutation AGUFin major is responsible for 98% of the AGUFin alleles. Another mutation AGUFin minor has been identified in eight compound heterozygote patients who have AGUFin major mutation in their other allele. In addition four compound heterozygote patients have AGUFin major in one allele and unknown AGUFin mutation in the other allele. To study the origin of these mutations the haplotype analysis was performed on six patients with AGUFin minor mutation and four patients with unknown AGUFin mutation using nine microsatellite markers on the 7.6 cM chromosome region on 4q28-4qter. The haplotype data suggest that one founder mutation is responsible of all AGUFin minor alleles. Allelic association was also observed in AGUFin major chromosomes. Patients with unknown mutation did not share a common haplotype and therefore most likely have different origin.     

Molecular Pathogenesis of Wilson Disease Among Indians: A Perspective on Mutation Spectrum in ATP7B gene, Prevalent Defects, Clinical Heterogeneity and Implication Towards Diagnosis

Aims We aim to identify the molecular defects in the ATP7B, the causal gene for Wilson disease (WD), in eastern Indian patients and attempt to assess the overall mutation spectrum in India for detection of mutant allele for diagnostic purposes. Methods Patients from 109 unrelated families and their first-degree relatives comprising 400 individuals were enrolled in this study as part of an ongoing project. Genomic DNA was prepared from the peripheral blood of Indian WD patients. PCR was done to amplify the exons and flanking regions of the WD gene followed by sequencing, to identify the nucleotide variants. Results In addition to previous reports, we recently identified eight mutations including three novel (c.3412 + 1G > A, c.1771 G > A, c.3091 A > G) variants, and identified patients with variable phenotype despite similar mutation background suggesting potential role of modifier locus. Conclusions So far we have identified 17 mutations in eastern India including five common mutations that account for 44% of patients. Comparative study on WD mutations between different regions of India suggests high genetic heterogeneity and the absence of a single or a limited number of common founder mutations. Genotype–phenotype correlation revealed that no particular phenotype could be assigned to a particular mutation and even same set of mutations in different patients showed different phenotypes.     

GM2-gangliosidosis B1 variant: analysis of beta-hexosaminidase alpha gene abnormalities in seven patients

A single nucleotide transition within exon 5 of the beta-hexosaminidase alpha chain gene was identified in a Puerto Rican patient with GM2-gangliosidosis B1 variant as the mutation responsible for the unusual enzymological characteristics of this variant (G533----A; Arg178----His) (the DN-allele). A total of seven patients with enzymological characteristics of B1 variant have since been studied. They were Puerto Rican (DN), Italian, French, Spanish, two patients of mixed ethnic origin (English/Italian/Hungarian and English/French/Azores), and a Czechoslovakian. In confirmation of our earlier finding based on screening with allele-specific probes, all patients except the one from Czechoslovakia carried the same DN-allele. A new point mutation found in this patient changed the same codon affected in the DN-allele (C532----T; Arg178----Cys). An asymptomatic Japanese individual included as a control also carried one allele with the DN-mutation. Site-directed mutagenesis and expression studies in COS I cells demonstrated that either of the two point mutations abolishes the catalytic activity of the alpha subunit. The Spanish patient was homozygous for the DN-allele, but others were all compound heterozygotes. The Puerto Rican patient was a compound heterozygote with the DN-mutation in one allele and with the four-base insertion in exon 11, one of the two mutations found in the classical Ashkenazi Jewish Tay-Sachs disease, in the other allele. Abnormalities of the other allele were not identified in all other compound heterozygous patients. In these patients, the level of mRNA derived from the other allele was variable, ranging from being undetectable to being much lower than normal. This series of studies uncovered a new B1 variant mutation, confirmed our preliminary finding that the DN-allele has a surprisingly wide geographic and ethnic distribution, and pointed out the highly complex nature of the molecular genetics of this rare disorder. They also support our working hypothesis that mutations responsible for the unique enzymological characteristics of the B1 variant should be located in or near exon 5 of the gene and that this region of the enzyme protein is critical for its catalytic function.     

A Novel Tandem Mass Spectrometry Method for Rapid Confirmation of Medium- and Very Long-Chain acyl-CoA Dehydrogenase Deficiency in Newborns

Background

Newborn screening for medium- and very long-chain acyl-CoA dehydrogenase (MCAD and VLCAD, respectively) deficiency, using acylcarnitine profiling with tandem mass spectrometry, has increased the number of patients with fatty acid oxidation disorders due to the identification of additional milder, and so far silent, phenotypes. However, especially for VLCADD, the acylcarnitine profile can not constitute the sole parameter in order to reliably confirm disease. Therefore, we developed a new liquid chromatography tandem mass spectrometry (LC-MS/MS) method to rapidly determine both MCAD- and/or VLCAD-activity in human lymphocytes in order to confirm diagnosis.

Methodology

LC-MS/MS was used to measure MCAD- or VLCAD-catalyzed production of enoyl-CoA and hydroxyacyl-CoA, in human lymphocytes.

Principal Findings

VLCAD activity in controls was 6.95±0.42 mU/mg (range 1.95 to 11.91 mU/mg). Residual VLCAD activity of 4 patients with confirmed VLCAD-deficiency was between 0.3 and 1.1%. Heterozygous ACADVL mutation carriers showed residual VLCAD activities of 23.7 to 54.2%. MCAD activity in controls was 2.38±0.18 mU/mg. In total, 28 patients with suspected MCAD-deficiency were assayed. Nearly all patients with residual MCAD activities below 2.5% were homozygous 985A>G carriers. MCAD-deficient patients with one other than the 985A>G mutation had higher MCAD residual activities, ranging from 5.7 to 13.9%. All patients with the 199T>C mutation had residual activities above 10%.

Conclusions

Our newly developed LC-MS/MS method is able to provide ample sensitivity to correctly and rapidly determine MCAD and VLCAD residual activity in human lymphocytes. Importantly, based on measured MCAD residual activities in correlation with genotype, new insights were obtained on the expected clinical phenotype.     

Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population in Jordan

Background Coagulation factor II G20210A and coagulation factor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Methods and Results factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR?CRFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5?% and an allele frequency of 1.25?%. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3?% and allele frequency of 9.2?%. No homozygous allele was found. Conclusion Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1?C6?%) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2?C15?%).     

Association of the widespread A149P hereditary fructose intolerance mutation with newly identified sequence polymorphisms in the aldolase B gene.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease resulting from the catalytic deficiency of fructose 1-phosphate aldolase (aldolase B) in fructose-metabolizing tissues. The A149P mutation in exon 5 of the aldolase B gene, located on chromosome 9q21.3-q22.2, is widespread and the most common HFI mutation, accounting for 57% of HFI chromosomes. The possible origin of this mutation was studied by linkage to polymorphisms within the aldolase B gene. DNA fragments of the aldolase B gene containing the polymorphic marker loci from HFI patients homozygous for the A149P allele were amplified by PCR. Absolute linkage to a common PvuII RFLP allele was observed in 10 A149P homozygotes. In a more informative study, highly heterozygous polymorphisms were detected by direct sequence determination of a PCR-amplified aldolase B gene fragment. Two two-allele, single-base-pair polymorphisms, themselves in absolute linkage disequilibrium, in intron 8 (C at nucleotide 84 and A at nucleotide 105, or T at 84 and G at 105) of the aldolase B gene were identified. Mendelian segregation of these polymorphisms was confirmed in three families. Allele-specific oligonucleotide (ASO) hybridizations with probes for both sequence polymorphisms showed that 47% of 32 unrelated individuals were heterozygous at these loci; the calculated PIC value was .37. Finally, ASO hybridizations of PCR-amplified DNA from 15 HFI patients homozygous for the A149P allele with probes for these sequence polymorphisms revealed absolute linkage disequilibrium between the A149P mutation and the 84T/105G allele. These results are consistent with a single origin of the A149P allele and subsequent spread by genetic drift.     

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932–939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype–phenotype correlation of the disease.     

Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number.

The autosomal recessive neuromuscular disorder proximal spinal muscular atrophy (SMA) is caused by the loss or mutation of the survival motor neuron (SMN) gene, which exists in two nearly identical copies, telomeric SMN (telSMN) and centromeric SMN (cenSMN). Exon 7 of the telSMN gene is homozygously absent in approximately 95% of SMA patients, whereas loss of cenSMN does not cause SMA. We searched for other telSMN mutations among 23 SMA compound heterozygotes, using heteroduplex analysis. We identified telSMN mutations in 11 of these unrelated SMA-like individuals who carry a single copy of telSMN: these include two frameshift mutations (800ins11 and 542delGT) and three missense mutations (A2G, S262I, and T274I). The telSMN mutations identified to date cluster at the 3' end, in a region containing sites for SMN oligomerization and binding of Sm proteins. Interestingly, the novel A2G missense mutation occurs outside this conserved carboxy-terminal domain, closely upstream of an SIP1 (SMN-interacting protein 1) binding site. In three patients, the A2G mutation was found to be on the same allele as a rare polymorphism in the 5' UTR, providing evidence for a founder chromosome; Ag1-CA marker data also support evidence of an ancestral origin for the 800ins11 and 542delGT mutations. We note that telSMN missense mutations are associated with milder disease in our patients and that the severe type I SMA phenotype caused by frameshift mutations can be ameliorated by an increase in cenSMN gene copy number.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.